Carotenoids represent one of the most widely distributed and structurally diverse classes of natural pigments, producing pigment colors of light yellow to orange to deep red color. Eye-catching examples of carotenogenic tissues include carrots, tomatoes, red peppers, and the petals of daffodils and marigolds. Carotenoids are synthesized by all photosynthetic organisms, as well as some bacteria and fungi. These pigments have important functions in photosynthesis, nutrition, and protection against photooxidative damage. For example, animals do not have the ability to synthesize carotenoids but must obtain these nutritionally important compounds through their dietary sources.
Industrially, only a few carotenoids are used for food colors, animal feeds, pharmaceuticals, and cosmetics, despite the existence of more than 600 different carotenoids identified in nature. This is largely due to difficulties in production. Presently, most of the carotenoids used for industrial purposes are produced by chemical synthesis; however, these compounds are very difficult to make chemically (Nelis and Leenheer, Appl. Bacteriol. 70:181-191 (1991)). Natural carotenoids can either be obtained by extraction of plant material or by microbial synthesis; but, only a few plants are widely used for commercial carotenoid production and the productivity of carotenoid synthesis in these plants is relatively low. As a result, carotenoids produced from these plants are very expensive. One way to increase the productive capacity of biosynthesis would be to apply recombinant DNA technology (reviewed in Misawa and Shimada, J. Biotech. 59:169-181 (1998)). Thus, it would be desirable to produce carotenoids in non-carotenogenic bacteria and yeasts, thereby permitting control over quality, quantity and selection of the most suitable and efficient producer organisms. The latter is especially important for commercial production economics (and therefore availability) to consumers.
Structurally, the most common carotenoids are 40-carbon (C40) terpenoids; however, carotenoids with only 30 carbon atoms (C30; diapocarotenoids) are detected in some species. Biosynthesis of each of these types of carotenoids are derived from the isoprene biosynthetic pathway and its five-carbon universal isoprene building block, isopentenyl pyrophosphate (IPP). This biosynthetic pathway can be divided into two portions: 1) the upper isoprene pathway, which leads to the formation of farnesyl pyrophosphate (FPP); and 2) the lower carotenoid biosynthetic pathway, comprising various crt genes which convert FPP into long C30 and C40 carotenogenic compounds. Both portions of this pathway are shown in FIG. 1.
Typically, the formation of phytoene represents the first step unique to biosynthesis of C40 carotenoids (FIGS. 1 and 2). Phytoene itself is a colorless carotenoid and occurs via isomerization of IPP to dimethylallyl pyrophosphate (DMAPP) by isopentenyl pyrophosphate isomerase. The reaction is followed by a sequence of 3 prenyltransferase reactions in which geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP) are formed. The gene crtE, encoding GGPP synthetase, is responsible for this latter reaction. Finally, two molecules of GGPP condense to form phytoene (PPPP). This reaction is catalyzed by phytoene synthase (encoded by the gene crtB).
Lycopene is the first “colored” carotenoid produced from phytoene. Lycopene imparts the characteristic red color of ripe tomatoes and has great utility as a food colorant. It is also an intermediate in the biosynthesis of other carotenoids in some bacteria, fungi and green plants. Lycopene is prepared biosynthetically from phytoene through four sequential dehydrogenation reactions by the removal of eight atoms of hydrogen, catalyzed by the gene crtl (encoding phytoene desaturase). Intermediates in this reaction are phytofluene, ζ-carotene, and neurosporene.
Lycopene cyclase (CrtY) converts lycopene to β-carotene, the second “colored” carotenoid. β-carotene is a typical carotene with a color spectrum ranging from yellow to orange. Its utility is as a colorant for margarine and butter, as a source for vitamin A production, and recently as a compound with potential preventative effects against certain kinds of cancers.
β-carotene is converted to zeaxanthin via a hydroxylation reaction resulting from the activity of β-carotene hydroxylase (encoded by the crtZ gene). For example, it is the yellow pigment that is present in the seeds of maize. Zeaxanthin is contained in feeds for hen or colored carp and is an important pigment source for their coloration. Finally, zeaxanthin can be converted to zeaxanthin-β-monoglucoside and zeaxanthin-β-diglucoside. This reaction is catalyzed by zeaxanthin glucosyl transferase (encoded by the crtX gene).
In addition to the carotenoid biosynthetic genes and enzymes responsible for creation of phytoene, lycopene, β-carotene, zeaxanthin, and zeaxanthin-β-glucosides, various other crt genes are known which enable the intramolecular conversion of C40 compounds to produce numerous other functionalized carotenoid compounds by: (i) hydrogenation, (ii) dehydrogenation, (iii) cyclization, (iv) oxidation, (v) esterification/ glycosylation, or any combination of these processes.
One organism capable of C40 carotenoid synthesis and a potential source of crt genes is Pectobacterium cypripedii (formerly classified as Erwinia cypripedii). The genus Erwinia has undergone substantial examination and reclassification within the last few decades. Previously, Dye had classified the members of the genus Erwinia into four natural clusters, consisting of the “carotovora group” (N. Z. J. Sci. 12:81-97 (1969)), the “amylovora group” (N. Z. J. Sci. 11:590-607 (1968)), the “herbicola group” (N. Z. J. Sci. 12:223-236 (1969)) and the “atypical Erwinias” (N. Z. J. Sci. 12:833-839 (1969)). This categorization was basically supported in Kwon et al. (Inter. J. System. Bacteriol. 47(4):1061-1067 (1997)), a study which utilized the 16S rDNA sequences of sixteen Erwinia species as a mechanism for phylogenetic analysis. And, most recently, Hauben et al. (Syst. Appl. Microbiol. 21(3):384-397 (August 1998)) examined the 16S rDNA sequences of twenty-nine species of the genera Erwinia, Pantoea and Enterobacter, and compared these sequences with those of other members of the Enterobacteriaceae. As with the work of Dye (supra) and Kwon et al. (supra), Hauben et al. also determined that species within the large former genus Erwinia may be divided into four phylogenetic groups, as shown below:                Cluster I comprises Erwinia amylovora, E. mallotivora, E. persicinus, E. psidii, E. rhapontici and E. tracheiphila;         Cluster II comprises Pectobacterium carotovorum subsp. atrosepticum comb. nov., P. carotovorum subsp. betavasculorum comb. nov., P. carotovorum subsp. carotovorum comb. nov., P. carotovorum subsp. odoriferum comb. nov., P. carotovorum subsp. wasabiae comb. nov., P. cacticidum comb. nov., P. chrysanthemi and P. cypripedii;         Cluster III comprises organisms within the new genus Brenneria gen. nov., which are identified respectively as B. alni comb. nov., B. nigrifluens, comb. nov., B. paradisiaca comb. nov., B. quercina comb. nov., B. rubrifaciens comb. nov. and B. salicis comb. nov.; and        Cluster IV comprise the species of the genus Pantoea (e.g., Pantoea stewartii subsp. stewartii (formerly Erwinia stewartii), P. agglomerans (formerly Erwinia herbicola), and P. ananatis (formerly Erwinia uredovora)).Despite lack of agreement between Hauben et al. (supra) and Kwon et al. (supra) concerning the species most closely related to Pectobacterium cypripedii, both studies clearly recognize that this organism is in a distinct cluster separate from those organisms originally recognized by Dye (supra) as the “herbicola group” and currently classified by Hauben et al. (supra) as Cluster IV “Pantoea” organisms.        
Numerous studies have examined carotenoid biosynthesis within members of Cluster IV (according to Hauben et al., supra) of this broad group of bacteria all formerly known within the genus Erwinia. For example, several reviews discuss the genetics of carotenoid pigment biosynthesis, such as those of G. Armstrong (J. Bact. 176: 4795-4802 (1994); Annu. Rev. Microbiol. 51:629-659 (1997)). And, gene sequences encoding crtEXYIBZ are available for Pantoea agglomerans (formerly known as E. herbicola EHO-10 (ATCC #39368)), P. ananatis (formerly known as E. uredovora 20D3 (ATCC #19321)), P. stewartii (formerly known as E. stewartii (ATCC #8200)), and P. agglomerans pv. milletiae (U.S. Pat. Nos. 5,656,472; 5,5545,816; 5,530,189; 5,530,188; 5,429,939; WO 02/079395 A2; see also GenBank® Accession Nos. M87280, D90087, AY166713, AB076662; respectively).
However, genes involved in the carotenoid biosynthetic pathway from organisms classified in Cluster I, II, and/or III (as defined by Hauben et al., supra) of this diverse group of organisms are not described in the existing literature. The problem to be solved, therefore, is to identify nucleic acid sequences encoding all or a portion of the carotenoid biosynthetic enzymes from organisms classified within these clusters to facilitate studies to better understand carotenoid biosynthetic pathways, provide genetic tools for the manipulation of those pathways, and provide a means to synthesize carotenoids in large amounts by introducing and expressing the appropriate gene(s) in an appropriate host. This will lead to carotenoid production superior to synthetic methods.
Applicants have solved the stated problem by isolating six unique open reading frames (ORFs) encoding enzymes in the carotenoid biosynthetic pathway from a yellow-pigmented bacterium classified as Pectobacterium cypripedii strain DC416. This organism represents Cluster II (as defined by Hauben et al., supra) of the revised phylogenetic group of organisms all formerly known within the genus Erwinia. 